Characterization of FcXTH2, a Novel Xyloglucan Endotransglycosylase/Hydrolase Enzyme of Chilean Strawberry with Hydrolase Activity.

Xyloglucan endotransglycosylase/hydrolases (XTHs) are cell wall enzymes with hydrolase (XEH) and/or endotransglycosylase (XET) activities. As they are involved in the modification of the xyloglucans, a type of hemicellulose present in the cell wall, they are believed to be very important in different processes, including growth, development, and fruit ripening. Previous studies suggest that XTHs might play a key role in development and ripening of Fragaria chiloensis fruit, and its characterization is pending. Therefore, in order to provide a biochemical characterization of the FcXTH2 enzyme to explain its possible role in strawberry development, the molecular cloning and the heterologous expression of FcXTH2 were performed. The recombinant FcXTH2 was active and displayed mainly XEH activity. The optimal pH and temperature are 5.5 and 37 °C, respectively. A KM value of 0.029 mg mL-1 was determined. Additionally, its protein structural model was built through comparative modeling methodology. The model showed a typically ß-jelly-roll type folding in which the catalytic motif was oriented towards the FcXTH2 central cavity. Using molecular docking, protein-ligand interactions were explored, finding better interaction with xyloglucan than with cellulose. The data provided groundwork for understanding, at a molecular level, the enzymatic mechanism of FcXTH2, an important enzyme acting during the development of the Chilean strawberry.

Immune Modulation Ability of Hepcidin from Teleost Fish.

Antimicrobial peptides (AMP) play an essential role in the innate immune system, modulating the defense response. In a previous study, we demonstrated the antimicrobial activity of synthetic hepcidin (hep20) from rainbow trout (Oncorhynchus mykiss), and its protective effect in European sea bass (Dicentrarchus labrax) challenged with Vibrio anguillarum. Additionally, we described the uptake and distribution of hep20 in different tissues and leukocyte cells. Interestingly, various AMPs characterized in high vertebrates, called host defense peptides (HDPs), also possess immunomodulation activity. For that reason, the present study explores the immunomodulatory abilities of hep20 through in vitro and in vivo studies. First, a monocyte/macrophage RTS-11 cell line from rainbow trout was used to evaluate hep20 effects on pro- and anti-inflammatory cytokines in fish leukocyte cells. Next, the European sea bass juveniles were used to determine if hep20 can regulate the expression of cytokines in fish immune tissues. The results show that hep20 was uptake inner to RTS-11 cells and was able to induce the expression of IL-10, IL-1ß, and TNFα at transcriptional and protein levels. Then, the European sea bass juveniles were given intraperitoneal injections of the peptide. At 1, 3, 7, 14, and 21 days post-injection (dpi), IL-10, IL -1ß, and TNFα mRNA were quantified in the anterior gut, spleen, and head kidney. The hep20 was able to up-regulate cytokine gene expression in these tissues, mainly in the head kidney. Furthermore, the evaluated cytokines showed a cyclical tendency of higher to lesser expression. Finally, a bioinformatics analysis showed that the structure adopted by hep20 is similar to the γ-core domain described for cysteine-stabilized AMP, defined as immunomodulatory and antimicrobial, which could explain the ability of hep20 to regulate the cytokine expression. This study provides new insights into immunomodulatory function complementary to the previously established antimicrobial activity of hep20, suggesting a role as an HDP in teleost fish. These facts are likely to be associated with molecular functions underpinning the protective effect of fish hepcidin against pathogens.

Molecular insights of a xyloglucan endo-transglycosylase/hydrolase of radiata pine (PrXTH1) expressed in response to inclination: Kinetics and computational study.

Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies confirmed XET activity for PrXTH1 protein from radiata pine. XTHs could interact with many hemicellulose substrates, but the favorite substrate of PrXTH1 is still unknown. The prediction of union type and energy stability of the complexes formed between PrXTH1 and different substrates (XXXGXXXG, XXFGXXFG, XLFGXLFG and cellulose) were determined using bioinformatics tools. Molecular Docking, Molecular Dynamics, MM-GBSA and Electrostatic Potential Calculations were employed to predict the binding modes, free energies of interaction and the distribution of electrostatic charge. The results suggest that the enzyme formed more stable complexes with hemicellulose substrates than cellulose, and the best ligand was the xyloglucan XLFGXLFG (free energy of -58.83 ±â€¯0.8 kcal mol-1). During molecular dynamics trajectories, hemicellulose fibers showed greater stability than cellulose. Aditionally, the kinetic properties of PrXTH1 enzyme were determined. The recombinant protein was active and showed an optimal pH 5.0 and optimal temperature of 37 °C. A Km value of 20.9 mM was determined for xyloglucan oligomer. PrXTH1 is able to interact with different xyloglycans structures but no activity was observed for cellulose as substrate, remodeling cell wall structure in response to inclination.

Glycosylation is important for FcXTH1 activity as judged by its structural and biochemical characterization.

Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies suggest that XTHs might play a key role in ripening of Fragaria chiloensis fruit as FcXTH1 transcripts increase as fruit softens. FcXTH1 protein sequence contains a conserved N-glycosylation site adjacent to catalytic residues. The FcXTH1 structure was built through comparative modeling methodology, the structure displays a ß-jellyroll-type folding with a curvature generated by eight antiparallel ß-sheets that holds the catalytic motif that is oriented towards the central cavity of the protein. Through Molecular Dynamic Simulations (MDS) analyses the protein-ligand interactions of FcXTH1 were explored, finding a better interaction with xyloglucans than cellulose. Nevertheless, the stability of the protein-ligand complex depends on the glycosylation state of FcXTH1: better energy interactions were determined for the glycosylated protein. As a complement, the molecular cloning and heterologous expression of FcXTH1 in Pichia pastoris was performed, and the recombinant protein was active and displayed strict XET activity. A KM value of 17.0 µM was determined for xyloglucan oligomer. The deglycosylation of FcXTH1 by PNGase-F treatment affects its biochemical properties (increase KM and reduce kcat/KM ratio) and reduces its stability. As a conclusion, glycosylation of FcXTH1 is important for its biological function.

Perfil clínico-epidemiológico de pacientes con sospecha diagnóstica de glucogenosis hepática de 1 a 18 años en el Hospital Nacional de Niños Benjamín Bloom en el período 2005 a 2015 / Clinical-epidemiological profile of patients with suspected diagnosis of hepatic glycogenosis aged 1 to 18 years at the Benjamín Bloom National Children's Hospital in the period 2005 to 2015

Las glucogenosis son alteraciones del metabolismo del glucógeno caracterizados por la existencia de depósitos cuantitativa o cualitativamente anormales de este polisacárido en el organismo. La hipoglicemia es el hecho característico y que define muchas veces per se la enfermedad. El objetivo es describir los signos, síntomas, hallazgos de laboratorio y gabinete que presentaron los pacientes, y que orientaron al especialista al diagnóstico de una enfermedad de depósito del glucógeno. Se realizó un estudio transversal, retrospectivo y descriptivo. La población fue de 20 pacientes obtenidos de los registros de la consulta externa de Gastroenterología con primer diagnóstico de Enfermedades de depósito de glucógeno, utilizando el instrumento de recolección. Las tablas y gráficos se realizaron utilizando el programa Microsoft Excel. La edad promedio al momento del diagnóstico fue de 4.55+/-2.60, siendo más frecuente las edades de 6 a 10 años con 9 pacientes (45%). De los 20 pacientes, 15 (75%) fueron del género masculino teniendo una relación M/F de 3:1. En los resultados de laboratorio se encontró en 17 (85%) alteraciones de transaminasas y, en los de gabinete, a 20 (100%) se les realizó ultrasonografía abdominal con hepatomegalia como hallazgo más frecuente. Se le practicó biopsia hepática a 17 (85%) pacientes y el hallazgo más encontrado fue el aumento del depósito de glucógeno. Conclusiones: En los pacientes se observa predominio del sexo masculino. El signo clínico que ha presentado la mayoría ha sido la hepatomegalia con distensión abdominal y el que inició el estudio de estos pacientes. La ultrasonografía fue el único estudio de gabinete utilizado. La biopsia hepática contribuyó al diagnóstico al describir depósitos de glucógeno como hallazgo